Request for Research Supplement for R01 AG055357 through the PA-18-591 mechanism Abstract for Supplement of R01 AG055357 ?Biochemical and functional interactions of a-actinin and calmodulin with Cav1.2? Our long-term interest is to determine the molecular mechanisms that regulate the L-type channel Cav1.2 in health and disease (e.g., Science 293, 98; Science 293, 2205; PNAS 103, 7500; Neuron 78, 483). We found that a-actinin binds directly to the IQ motif of Cav1.2 and augments its surface localization (Neuron 78, 483). Supported by the parental R01 AG055357 grant we show through structural analysis, which informed point mutations in the IQ motif, that binding of a-actinin to the IQ motif strongly augments channel open probability (Po). Aim 1 is a comprehensive analysis of trafficking kinetics of WT and point mutated Cav1.2. Aim 2 is to determine current density, gating currents, and single channel currents for a-actinin binding - deficient point mutations of Cav1.2. Our structural analysis has guided charge reversal experiments for unequivocal assignment of deficits in surface expression and Po in the Cav1.2 mutants to loss of a-actinin binding (under full review by Neuron). We also found that Ca2+ influx through Cav1.2 leads to displacement of a-actinin from the IQ motif by Ca2+/calmodulin and in parallel to endocytosis of Cav1.2 (readied for publication). Aim 3 is to test the role of a-actinin in Ca2+ influx into spines and regulation of gene expression via NFAT, whether b- amyloid peptide 1-42 augments Po of CaV1.2 by stimulating the CaV1.2-associated b2 adrenergic receptor and thereby S1928 phosphorylation via PKA, and whether a-actinin association of Cav1.2 is increased in rodent models of senility and Alzheimer?s disease (AD). Increased Cav1.2 channel activity contributes to senile symptoms and AD (e.g., Science 272, 1017; Science 243, 809). Thus our work on the functional interplay of Cav1.2 with a-actinin and calmodulin is of high significance for understanding and ultimately development of treatments of these brain diseases. On a broader perspective it is of physiological importance as it will define important aspects that govern the functional availability of Cav1.2 with its manifold functions in neurons and beyond including learning and memory. The Aim for the request of a Supplement is to define by Cyro-electron microscopy the structure of CaV1.2 in complex with a-actinin obtained from healthy rats and compare with the CaV1.2/a-actinin structure isolated from B6.Cg-Tg(APP695)3Dbo Tg(PSEN1dE9)S9Dbo/Mmjax mice. Rationale: Consistent with our finding that surface expression of CaV1.2 increases during aging in rats (Aging Cell 13, 111-120), we found that a-actinin binding to CaV1.2 is also strongly increased in aging rats. Thus, during aging the propensity of CaV1.2 to bind to a-actinin is augmented, which must be due to conformational changes possibly because of alterations in the splicing of the channel mRNA or its phosphorylation. We will determine the structural changes that mediate increased a-actinin binding to CaV1.2.